5 Nucleus with DNA 2n = 18 Total size carrot genome: 473 MbpZoom in on nucleus 9 different chromosomesDiploid species, 2 copies of each chromosome, one from mother and one from father, 18 in totalZoom in on chromosome, folded complex of proteins and DNADNA forms a double helix, consists of two separate strands of nucleotides.The nucleotides form basepairs connecting the two strands.4 different nucleotides: A, T, C en G. The two strands are complementary to each other, since A always pairs with T and C always with G.The order of nucleotides is called a sequence and this encodes genetic information.The total size of the carrot genome is 473 MbpTotal size carrot genome:473 Mbp

6 The plant Within a plant, all plant cells contain the same DNAFor plant growth, cells need to dividePrior to cell division → DNA duplication

7 DNA duplication Double stranded DNA Separation of two strandsIncorporation ofnucleotidesSeparation of two strandsTwo copies ofdouble stranded DNADouble stranded DNAThe first step is to separate the two strandsSince the strands are complementary, they can both act as template for two new strands.The correct nucleotides are linked one by one to the new strandsAfter duplication is finished, there are two exact copies of the original ds DNA

8 2 identical diploïd cellsCell DivisionAfter duplication has finished, the cell can divide itself and both cells still have the same DNA content. In this way all plant cells are identical for their DNA content.DNA duplicationMitosis2 identical diploïd cells

9 Production of gametes DNA duplication Meiosis 4 unique haploïd gametesAlso during the production of the gametes (eggcells and pollen grains) the first step is DNA duplication. After DNA duplication, the 2 copies of each chromosome lign up and a crossover may occur: two strands break at the same position and reconnect to the other copy. The two copies are divided over two different cells, which are again divided over two cells. This results in 4 haploid gametes with or without crossing-overs. On average their will be one crossing-over per chromosome.DNA duplicationMeiosis4 uniquehaploïd gametes

10 SummaryA plant consists of many different cells, each with identical DNA contentDNA consists of two complementary strandsDuring duplication, each strand acts as a template to produce two identical copiesDuplication of DNA occurs prior to:Cell division → two identical diploïd cellsGamete production → four unique haploïd cells

11 PCR Polymerase Chain Reaction Amplification of DNAMimics the process of DNA duplication in the plantThe polymerase (enzyme) requires dsDNA to start building in nucleotidesThe DNA duplication can also be done in the lab

12 PCR denaturation annealing elongationUsually only a small region of interest needs to be amplifiedAfter denaturation DNA is single strandedpolymerase can only elongate ds DNA, we need a starting point.by adding small artificial pieces of DNA called primer with identical sequence to the region of interest,In order to make the primers, we need to know the sequence of the region of interestelongation

14 Molecular Markers Amplify a specific region of the genome through PCRVisualize the amplified fragmentPerform this step for different parent linesWhen an amplified fragment is different for two parent lines it becomes a molecular markerIf we have the sequence, we can make specific primers to amplify the specific region

28 Mapping a trait Errors in scoring make the analysis more complexBetter phenotyping results in better mappingEasy for traits determined by a single gene: one locus on the genome

29 Mapping a trait For complex traits use QTL mappingQTLs: Quantative Trait LociGenotyping and phenotyping a populationUse computer software to statistically calculate the positions on the genome (loci) that have an influence on the trait.

31 Advantages for carrot breedingMolecular markers provide an additional tool for breeders to select for their traits of interest.Selection with markers can be done at any stage with any part of the plantNew possibilities to combine traits of interest to create even better carrots